The goal of this proposal is to develop instrumentation for 3-D tomographic imaging of whole cells, and macromolecular assemblies in cells, at high-resolution (40-50 nm) using soft X -ray microscopy. This technology will bridge the existing gap between light and electron microscopy. To achieve this goal, three parallel efforts must be pursued: 1) A rapid freezing facility for specimens must be developed; 2) Computer controlled cryogenic sample stages for the x-ray microscope must be built; and 3) Tomographic analysis software must be installed and linked to the acquisition system. Ultimately, we will develop techniques and technology required for performing correlated light and x-ray microscopy on the same specimen. The development of this technology will provide a unique approach for examining fluorescently tagged proteins (i.e. constructs containing variants of green fluorescent protein) in live cells and then examining those same cells at high resolution in the X -ray microscope. Once completed our developments will be made available to the cell, molecular, and developmental biology community. In addition, the steady progress in high repetition rate, high pulse energy laser sources will enable taking this technology to individual laboratories in the near future. This will provide a powerful tool for structure-function analyses of proteins within cells.